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The Journal of Immunology, 2002, 169: 1705-1712.
Copyright © 2002 by The American Association of Immunologists

TCR/CD3 Down-Modulation and {zeta} Degradation Are Regulated by ZAP-701

Céline Dumont*, Nicolas Blanchard*, Vincenzo Di Bartolo{dagger}, Nathalie Lezot*, Evelyne Dufour{dagger}, Sébastien Jauliac{ddagger} and Claire Hivroz2,*

* Institut National de la Santé et de la Recherche Médicale, Unité 520, Institut Curie, and {dagger} Molecular Immunology Unit, Institut Pasteur, Paris, France; and {ddagger} Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA 02215


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
TCR down-modulation following binding to MHC/peptide complexes is considered to be instrumental for T cell activation because it allows serial triggering of receptors and the desensitization of stimulated cells. We studied CD3/TCR down-modulation and {zeta} degradation in T cells from two ZAP-70-immunodeficient patients. We show that, at high occupancy of the TCR, down-modulation of the CD3/TCR is comparable whether T cells express or do not express ZAP-70. However, if TCR occupancy was low, we found that CD3/TCR was down-regulated to a lesser extent in ZAP-70-negative than in ZAP-70-positive T cells. We studied CD3/TCR down-modulation in P116 (a ZAP-70-negative Jurkat cell-derived clone) and in P116 transfected with genes encoding the wild-type or a kinase-dead form of ZAP-70. Down-modulation of the TCR at high occupancy did not require ZAP-70, whereas at low TCR occupancy down-modulation was markedly reduced in the absence of ZAP-70 and in cells expressing a dead kinase mutant of ZAP-70. Thus, the presence of ZAP-70 alone is not sufficient for down-modulation; the kinase activity of this molecule is also required. The degradation of {zeta} induced by TCR triggering is also severely impaired in T cells from ZAP-70-deficient patients, P116 cells, and P116 cells expressing a kinase-dead form of ZAP-70. This defect in TCR-induced {zeta} degradation is observed at low and high levels of TCR occupancy. Our results identify ZAP-70, a tyrosine kinase known to be crucial for T cell activation, as a key player in TCR down-modulation and {zeta} degradation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
T cells are activated following the recognition by the TCR of antigenic peptides bound to MHC molecules. TCR is a multimeric protein complex consisting of the clonotypic {alpha}{beta} heterodimer, the CD3 {gamma}{delta}{epsilon} chains, and the {zeta} homodimer (1). The {alpha}{beta} heterodimer is responsible for specific recognition of the Ags, whereas the associated CD3 and {zeta} homodimer are responsible for signaling by the complex (2).

Like many other cell surface receptors, TCR-CD3-{zeta} complexes are constitutively internalized and recycled back to cell surface (3, 4, 5). It was shown in the early 1980s that the activation of T cells by Ag-loaded APCs, or mAbs directed against the TCR/CD3 complex, results in the down-modulation of TCR-CD3-{zeta} expression at the cell surface (Refs. 6 and 7 and reviewed in Ref. 8). This down-modulation may contribute to several features of the T cell response. The down-modulation of these complexes, by reducing the number of receptors at the cell surface, abolishes sustained signaling in T-APC conjugates and affects the responsiveness of T cells to further antigenic stimulation (6, 9). TCR down-modulation may also facilitate the serial engagement of many TCRs by a small number of TCR/peptide-MHC complexes (10).

The antigenic stimulation of T cells leads to activation of two protein tyrosine kinases (PTKs)3 of the src family: p56Lck and p59Fyn. It also leads to phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs), which are present in all the chains of the CD3-{zeta} complex. This, in turn, leads to the recruitment of an otherwise cytosolic PTK, ZAP-70, which is absolutely required for T cell function (11, 12). Receptor down-regulation and degradation are common to many membrane receptors with associated or intrinsic PTK activity, and in many cases these processes have been shown to be controlled by tyrosine kinase activity (13). The role of PTKs in TCR internalization and trafficking has been investigated but remains unclear. Some authors have reported that PTK inhibitors block anti-CD3 Ab-induced TCR down-regulation (14, 15), whereas others observed no such effect (16). Moreover, Jurkat cell mutants lacking the p56Lck PTK or the regulatory tyrosine phosphatase CD45 down-regulate TCRs in response to anti-CD3 Ab or superantigens less efficiently than the parental cell line (15, 17).

Consistent with the possible role of signaling in TCR down-modulation, a clear correlation was recently found between the loss of surface receptor and the proportion of TCRs generating full signals (18, 19). Partial agonists and antagonists elicit patterns of early {zeta} phosphorylation different from those induced by agonists (20), resulting in the nonactivation of ZAP-70 and a lower level of CD3/TCR down-regulation (19, 21).

The precise intracellular fates of the various components of the complex after T cell stimulation by Ag or superantigen are unclear. It has been shown that TCR-CD3-{zeta} complexes are degraded (22, 23) and that, at least for the {zeta}-chain, this degradation may occur in the lysosomal compartment (24).

The Syk PTK, which belongs to the same family as the ZAP-70 PTK, has been shown to regulate the transport of the {gamma}-chain of the FcR to lysosomes (25). Because {gamma} and {zeta} are similar in structure, we thought that ZAP-70 might play a similar role in the targeting of {zeta} to lysosomal compartments.

Therefore, we investigated the potential role of ZAP-70 in TCR down-modulation and {zeta} degradation in two models: human T cells from patients presenting immunodeficiency due to a lack of ZAP-70 and the P116 ZAP-70-deficient Jurkat clone (26) expressing different forms of ZAP-70. We found that ZAP-70 was involved in the degradation of {zeta} and that this process was controlled by the kinase activity of ZAP-70.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of PBMC and blasts

PBMC and CD4+ T cells from control donors and two patients with ZAP-70 immunodeficiency, described elsewhere (27, 28), were obtained as previously described (29). Blasts were obtained from the ZAP-70-deficient patients and two healthy donors by stimulating PBMC with 10 ng/ml PMA (Sigma-Aldrich, St. Louis, MO) and 1 µg/ml ionomycin (Calbiochem, La Jolla, CA). The culture medium consisted of 45% AIMV (Life Technologies, Rockville, MD), 45% RPMI 1640 (Life Technologies), and 10% FCS (Life Technologies) supplemented with 2 mM glutamine and 50 µg/ml gentamicin (Life Technologies). Recombinant human IL-2 (Chiron, Emeryille, CA) was added to a concentration of 100 U/ml 2 days later. The resulting blasts were used after 7–9 days of activation.

Cell lines, Abs, and reagents

The Jurkat cell-derived ZAP-70/Syk-deficient P116 cell line was kindly provided by Dr. T. Abraham (Department of Immunology, Mayo Clinic, Rochester, NY) and was maintained as previously described (26). P116 cells were transfected with the gene encoding the wild-type form of ZAP-70 or the kinase-dead form, bearing the D461/N mutation, subcloned into a pSR{alpha}-puro vector, as previously described (30). Stable clones were obtained by culturing the cells in the presence of 10 µg/ml puromycin and maintained as previously described (30). The Burkitt B cell line Raji was maintained as previously described (23).

The Abs used in this work were as follows: mouse mAb 4G10 (anti-phosphotyrosine, IgG2a), mAb UCHT1 (anti-CD3{epsilon}, IgG1), PE-conjugated mAb SK7 (anti-CD3{epsilon}, IgG1; BD Biosciences, Mountain View, CA), FITC-conjugated mAb Leu12 (anti-CD19; BD Biosciences), PE-conjugated anti-TCR{alpha}{beta} mAb (IgG2b; Beckman Coulter, Fullerton, CA), PE-conjugated F(ab')2 donkey anti-mouse IgG (H and L chains; Jackson ImmunoResearch Laboratories, West Grove, PA), anti-{zeta}-chain mAb (IgG1; Santa Cruz Biotechnology, Santa Cruz, CA), anti-tubulin mAb (IgG1; Amersham Pharmacia Biotech, Little Chalfont, U.K.), mAb H68.4 (anti-transferrin receptor, IgG1), anti-phospho-p44/42 mitogen-activated protein kinase (MAPK) mAb (IgG1; New England Biolabs, Beverly, MA), and anti-ZAP-70 mAb (IgG2a; BD Transduction Laboratories, Lexington, KY). Staphylococcal enterotoxin E (SEE) was obtained from Toxin Technology (Sarasota, FL).

T cell activation

Short-term activation of T cell blasts or Jurkat cell clones by anti-CD3{epsilon} mAb was performed at 37°C in RPMI 1640. CD3/TCR modulation by anti-CD3{epsilon} mAb was induced by incubating the cells for the time indicated with several concentrations of purified UCHT1. For the activation by SEE, T cells were incubated for 1 h at 37°C in the presence of 10 µg/ml cycloheximide (Sigma-Aldrich) or left in normal medium, as stated in Results. They were then cultured at a 1:1 ratio with MHC class II+ Raji cells, which were pulsed for 1 h with various concentrations of SEE. The reaction was stopped by adding 0.1% sodium azide in PBS and the cells were lysed or stained for flow cytometry.

Analysis of surface CD3 and TCR analysis

Immunofluorescence and flow cytometry analyses were performed on a FACSCalibur flow cytometer (BD Biosciences). We acquired data from 5000 viable CD3+ cells, using a forward scatter/side scatter gate to select the cells that were alive. In experiments involving activation by UCHT1, cells were labeled with UCHT1 and a PE-conjugated anti-mouse Ig as the secondary Ab. If both Raji B cells and T cells were present, then cells were labeled by incubation with a PE-conjugated anti-CD3 mAb or a PE-conjugated anti-TCR{beta} mAb, together with an FITC-conjugated anti-CD19 mAb. CD3+ T cells were analyzed using a gate that excluded CD19+ cells. Results are expressed as a percentage of the mean fluorescence intensity (MFI) of control cells incubated without stimuli under identical conditions.

Immunoprecipitation and Western blot analysis

After activation, cells were lysed by incubation in lysis buffer (20 mM Tris-HCl (pH 7.4), 140 mM NaCl, 2 mM EDTA, 50 mM NaF, 1% Nonidet P-40, 0.5% NaDOC, 0.1% SDS, 100 µM Na3VO4, 10 µg/ml each of antipain, pepstatin, leupeptin, and aprotinin, and 1 mM PMSF) for 20 min at 4°C. Nuclei and cell debris were removed by centrifugation. Lysates were subjected to SDS-PAGE in reducing conditions, and the separated proteins were electroblotted onto Immobilon P membrane (Millipore, Bedford, MA). The Ab/Ag complexes were visualized by ECL detection according to the manufacturer’s instructions (Amersham Pharmacia Biotech).

Surface biotinylation and precipitation of biotinylated proteins

Jurkat cells were washed and resuspended in cold PBS at a density of 2 x 107 cells/ml. Biotin-X-NHS (Calbiochem) in PBS was added to a final concentration of 500 µg/ml and the cells were incubated for 7 min on ice. The cells were centrifuged and the resulting pellet was resuspended in 0.1 M glycine in PBS and incubated on ice for 10 min. Cell viability was not affected by biotinylation, as shown by trypan blue exclusion assays. Cells were left inactivated or were activated by incubation with Raji cells and SEE for 2.5 h, as described above. The cells were then lysed and biotinylated proteins were precipitated with streptavidin-conjugated agarose beads (Amersham Pharmacia Biotech). The recovered proteins were subjected to SDS-PAGE, electroblotted onto membranes, and detected by ECL.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Down-modulation of TCR and {zeta} degradation induced by TCR triggering are modified in T cells from ZAP-70-deficient patients

Following the activation of T cells via the TCR, TCR-CD3 complexes are rapidly down-regulated and the CD3-{zeta} chain is degraded (24).

We studied these phenomena in the T lymphocytes of two unrelated patients presenting ZAP-70 deficiencies. Neither of these patients has any ZAP-70 and both were described in previous studies (27, 28). CD4+ primary T cells and T cell blasts from patients 1 and 2 displayed levels of CD3 expression similar to those in CD4+ T cells or blasts from control donors (Fig. 1GoA). As previously reported, activation of the CD4+ T cells or T cell blasts of these two patients by anti-CD3{epsilon} mAb did not lead to extracellular signal-regulated kinase (Erk)-2 activation, Ca2+ mobilization, proliferation, or IL-2 production (27, 28). We used FACScan analysis to study the down-modulation of CD3 induced by activation with the anti-CD3 mAb UCHT1 in T cell blasts from a control donor and the two ZAP-70-deficient patients. At high concentrations of the anti-CD3 mAb UCHT1 (dilutions of 1/500–1/2500), 60–70% of the CD3 was down-regulated in blasts from the two ZAP-70-deficient patients and the control (Fig. 1GoB). For lower concentrations of anti-CD3 mAb (dilutions of 1/20,000 and 1/10,000), 15–20% of CD3 was down-regulated in the patients’ blasts, whereas 40–50% of the surface CD3 was down-modulated in control blasts.



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  		FIGURE 1. The CD3-induced down-modulation of CD3 and {zeta} degradation are altered in T cells from ZAP-70-deficient patients. A, CD3{epsilon} levels were determined by FACScan analysis in purified CD4+ T cells or T cell blasts from a control donor, patient 1, and patient 2. The shaded histograms correspond to the control isotype and the open histograms correspond to CD3-{epsilon} levels in cells. B, T cell blasts from the control donor or the two patients were incubated for 2.5 h at 37°C with several dilutions of UCHT1 ascites. Cells were then washed and resuspended in sodium azide in PBS at 4°C after which they were labeled with UCHT1 and a PE-conjugated anti-mouse IgG. CD3{epsilon} expression is expressed as a percentage of the MFI of resting cells ((MFI of activated T cells/MFI of resting T cells) x 100). C, CD3 activation does not induce {zeta} degradation in T cells from ZAP-70-deficient patients. Primary CD4+ T cells from patient 1 and a control donor (Ctl 1) were activated by incubation for 2 h with UCHT1 at a dilution of 1/1000 (left panel). T blasts from a control donor or patient 2 were activated with various dilutions of UCHT1. Total {zeta}-chain content was determined by Western blotting. Blots were also probed with an anti-tubulin mAb to check that an equal amount of protein was loaded in each lane.

 
Because down-regulation of the CD3/TCR complex has been shown to be followed by degradation (24), we studied degradation of the CD3-{zeta} chain after TCR activation. Primary CD4+ T cells and T cell blasts from a control donor and the patients were activated by incubation with UCHT1 for 1 h and lysed. Lysates were subjected to SDS-PAGE and the resolved proteins were electroblotted onto membranes. We probed the Western blot for {zeta} and tubulin, which was used as a control to check that equal amounts of protein were present in each lane. No degradation of CD3-{zeta} was observed in T cells from the patient (Fig. 1GoC). We performed similar experiments with blasts obtained from patient 2 and from control donors; no CD3-{zeta} degradation was observed in patient 2 blasts at any of the UCHT1 concentrations used (Fig. 1GoC). Thus, activation of the patient’s blasts by UCHT1 at dilutions of 10-3 and 4 x 10-4 did not induce {zeta} degradation, whereas, at these dilutions, the down-regulation of TCR/CD3 complexes was similar to that observed in normal T cell blasts.

These results suggest that ZAP-70 controls the TCR/CD3-mediated down-modulation of CD3 and the degradation of the CD3-{zeta} chain induced by TCR triggering.

TCR-induced down-modulation of CD3/TCR at low receptor occupancy depends on the presence and kinase activity of ZAP-70

To confirm the role of ZAP-70 in CD3/TCR down-modulation and CD3-{zeta} degradation, we studied these phenomena in another model: the P116 ZAP-70-deficient Jurkat cell clone (26). We used P116 or stable clones of P116 expressing the wild-type form of ZAP-70 (P116ZAPwt) or the D461/N kinase-dead form of ZAP-70 (P116ZAP{Delta}K) described elsewhere (30). We also used a wild-type Jurkat cell clone (clone 20) previously used in other down-regulation studies (31).

No ZAP-70 expression was observed in the P116 clone (Fig. 2Go). The two transfected clones, P116ZAPwt and P116ZAP{Delta}K, produced similar amounts of ZAP-70, more in each case than was produced by the wild-type Jurkat clone 20 (Fig. 2Go). We then checked the signaling properties of the various clones used in this study. We activated the cells by incubating them with the anti-CD3 mAb UCHT1. This led to the tyrosine phosphorylation of several proteins in clone 20 and P116ZAPwt, whereas only a few tyrosine phosphorylations were induced in P116 and P116ZAP{Delta}K. UCHT1 induced tyrosine phosphorylation of the kinase-dead ZAP-70 (Fig. 2Go, upper panel, *). We then studied the phosphorylation of Erk-1 and Erk-2 by immunoblotting with anti-phospho-MAPK mAb. UCHT1 induced the phosphorylation of Erk-1 and Erk-2 both in clone 20 and in P116ZAPwt (Fig. 2Go). UCHT1 activation of P116 also induced the phosphorylation of Erk-1 and Erk-2, albeit to a lesser extent than observed in clone 20 or P116ZAPwt. These results were surprising because we previously showed that UCHT1 did not induce MAPK phosphorylation in T cells from ZAP-70-deficient patients (28). No Erk phosphorylation was induced by UCHT1 in P116ZAP{Delta}K, suggesting that the kinase-dead form of ZAP-70 had a negative effect on the MAPK activation pathway. We then studied the phosphorylation of {zeta}. This chain was phosphorylated in all clones except the ZAP-70-deficient P116 cells. These results are similar to those of previous studies (26), including our own study showing that no {zeta} tyrosine phosphorylation was observed in anti-CD3 Ab-triggered T cells from ZAP-70-deficient patients (28). They probably reflect the fact that the association of ZAP-70 with phosphorylated {zeta} protects the ZAP-70 molecule from dephosphorylation (26). The amount of {zeta} expressed differed reproducibly between cell lines, with {zeta} levels highest in P116 cells, lowest in P116{Delta}K cells, and intermediate and similar in P116ZAPwt and clone 20 (Fig. 2Go, lower panel).



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  		FIGURE 2. Characterization of signaling defects in the P116-derived clones. T cells were left unactivated or stimulated with UCHT1 (ascites at a dilution of 10-3) for the time indicated and were then lysed. The upper panel shows a Western blot probed with the anti-phosphotyrosine mAb 4G10. The next two panels show a single blot probed with a ZAP-70-specific mAb and a phospho-MAPK-specific mAb, respectively. The last two panels show the phosphorylation of {zeta} on tyrosine residues and the total amount of {zeta}, sequentially detected by reprobing of the same blot.

 
For down-regulation experiments, T cells were first treated for 1 h with cycloheximide to prevent differences in results due to de novo synthesis of the CD3/TCR complex. Cells were then activated by incubation with various concentrations of the anti-CD3 mAb UCHT1 for 1 h in the presence of cycloheximide. CD3 down-modulation was measured by FACScan analysis. At high concentrations of UCHT1 (from 1.5 to 12.5 µg/ml), 40–50% of total CD3 was down-modulated in all the Jurkat clones tested (Fig. 3GoA). In contrast, at lower concentrations of UCHT1, the percentage of down-modulated CD3 in the P116 and in the P116ZAP{Delta}K was lower than in the wild-type Jurkat or the P116ZAPwt cells. Thus, under 0.1 µg/ml UCHT1, no down-modulation was observed in P116 or P116ZAP{Delta}K, whereas 10–20% of CD3 was down-modulated in the P116ZAPwt cells. Of note, the down-modulation induced by low concentrations of UCHT1 in wild-type Jurkat cells (clone 20) was intermediate between P116 or P116ZAP{Delta}K and P116ZAPwt cells. This could be due to the fact that P116ZAPwt cells express higher amounts of ZAP-70 than clone 20 cells as shown in Fig. 2Go.



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  		FIGURE 3. ZAP-70 controls the CD3 down-modulation induced at high levels of TCR occupancy. A wild-type Jurkat clone (clone 20), the parental P116 clone, and P116 expressing a gene encoding a wild-type or kinase-dead form of ZAP-70 (PZAPwt and PZAP{Delta}K, respectively) were treated for 1 h with cycloheximide. They were then stimulated by incubation for 1.5 h with serial dilutions of purified UCHT1 (A) or with Raji B cells pulsed with various concentrations of SEE (B). C, Cells were incubated with UCHT1 at 0.17 µg/ml and the reaction was stopped after various times for kinetic analysis of CD3 down-modulation. In all cases, cells were washed and resuspended in sodium azide in PBS at 4°C. They were labeled with UCHT1 and a PE-conjugated anti-mouse IgG. Surface expression of CD3 was assessed by FACScan analysis of triplicates and is expressed as a mean of percentages of unstimulated cells. SDs are shown.

 
We then activated the TCR with a more physiological activator to confirm that ZAP-70 does indeed control TCR/CD3 down-modulation. We used the Raji B cell line, which was pulsed in the presence or absence of various concentrations of the SEE superantigen. We began by studying TCR/CD3 down-modulation after activation of the various T cell lines by SEE. The T cells were first treated for 1 h with cycloheximide, which was also present throughout the activation. We observed down-regulation of CD3 (Fig. 3GoB) and TCR (data not shown), which was dependent on the concentration of SEE. As for anti-CD3 mAb-mediated stimulation, this down-modulation was different in Jurkat cells with a functional ZAP-70 and in P116 and P116ZAP{Delta}K cells. High doses of SEE (1–10-2 µg/ml) induced very similar levels of CD3 down-modulation in all the Jurkat cell-derived cell lines tested. In contrast, at low concentrations of SEE (10-3–10-5 µg/ml), the percentage of CD3 down modulated was lower in P116 and P116ZAP{Delta}K cells than in the P116ZAPwt clone or wild-type Jurkat cells. Of note, the number of conjugates formed between nonpulsed or SEE (0.1 µg/ml)-pulsed Raji B cells and all T cell lines used in this study were comparable (data not shown), showing that ZAP-70 did not control T cell adhesion to the APC in our model.

We then performed kinetic analysis of the down-regulation of CD3/TCR at low doses of UCHT1 (0.17 µg/ml). CD3 down-regulation was clearly less severe in the P116 and P116ZAP{Delta}K cell lines than in the other cell lines, at all time points tested (from 5 min to 2 h; Fig. 3GoC). Moreover, the down-modulation of CD3 was reproducibly delayed in these two cell lines. In the wild-type Jurkat clone (clone 20) and P116ZAPwt cell line, down-modulation was detected as early as 5 min after activation, whereas in P116 and P116ZAP{Delta}K cells it was observed only after 20 min of activation.

These results demonstrate that down-modulation of the CD3/TCR complex by CD3 activation is regulated by ZAP-70.

TCR-induced CD3-{zeta} degradation depends on the production and kinase activity of ZAP-70

We then studied CD3-{zeta} degradation after activation of the various clones with B cells pulsed with various concentrations of SEE. We performed these experiments on T cells previously treated for 1 h with cycloheximide. Cycloheximide was maintained in the medium throughout the activation to prevent the de novo synthesis of CD3-{zeta}. SEE induced the dose-dependent degradation of CD3-{zeta} in P116ZAPwt cells, some degradation at high doses only in P116 cells, and no degradation in P116ZAP{Delta}K cells, even at a concentration of 1 µg/ml (Fig. 4GoA). Degradation of CD3-{zeta} in the wild-type Jurkat was induced at all doses tested like in the P116ZAPwt. We then used densitometry to measure the intensity of the {zeta} band in each sample in five independent experiments. This quantitative analysis of {zeta} degradation confirmed that no {zeta} degradation was induced by the TCR activation of P116ZAP{Delta}K cells, even at high concentrations of superantigen (Fig. 4GoB). Defective degradation of {zeta} was observed after 3 h of activation by SEE (data not shown), showing that the lack of detection of degradation in these cells was not due to a delay in degradation. The results obtained for P116 cells were slightly different, in that some {zeta} degradation was observed at all concentrations of superantigen tested, although the level of degradation observed was always much lower than that in P116ZAPwt cells and in the wild-type Jurkat cells (clone 20). These differences between the results obtained with P116 and P116ZAP{Delta}K cells may reflect the greater extent to which TCR signaling is impaired in P116ZAP{Delta}K cells than in P116 cells (Fig. 2Go). Of note, although very similar, the dose-dependent degradation of {zeta} was reproducibly slightly less in clone 20 than in P116ZAPwt at all doses of SEE tested except the highest one (1 µg/ml). This could be due to the overexpression of ZAP-70 in P116ZAPwt shown in Fig. 2Go.



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  		FIGURE 4. ZAP-70 kinase activity is required for TCR-induced {zeta} degradation. Cells were treated for 1 h with cycloheximide; they were then stimulated for 2.5 h with Raji B cells pulsed with various concentrations of SEE and lysed after activation. The total level of {zeta} in each sample (5 x 105 cells/lane) was then analyzed by Western blotting with an anti-{zeta} mAb. A representative experiment is shown in A. Densitometric analysis of the {zeta} bands obtained in five independent experiments is shown in B and expressed as a percentage of the density obtained for nonactivated cells.

 
In the T cells of ZAP-70-deficient patients, there was a difference in the ZAP-70 requirements of {zeta} degradation and TCR/CD3 down-modulation because {zeta} degradation was low at both high and low levels of TCR occupancy, whereas TCR/CD3 down-modulation was inhibited only at low TCR occupancy.

Turnover of the {zeta}-chain is rapid in T cells (5, 32); therefore, we wanted to measure degradation of the pool of {zeta} that was once in the plasma membrane and was internalized constitutively or in response to TCR/CD3 triggering. We biotinylated P116, P116ZAPwt, and P116ZAP{Delta}K cells. Cells were lysed either immediately after biotinylation or after incubation at 37°C in medium alone, medium plus B cells, or medium plus B cells pulsed with SEE. Biotinylated proteins were precipitated with streptavidin-coupled agarose beads. The basal turnover of {zeta} in the various P116 clones was very similar (Fig. 5GoA). The percentage of biotinylated {zeta} degraded after 1.5 h of incubation at 37°C was 76% for P116, 70% for P116ZAPwt, and 70% for P116ZAP{Delta}K (mean of four independent experiments). These results strongly suggest that ZAP-70 is not involved in this degradation. In contrast, the degradation of biotinylated {zeta} induced by Raji pulsed with SEE was observed only in the P116ZAPwt cells, with no such degradation observed in the P116 and P116ZAP{Delta}K clones. We used the biotinylated transferrin receptors as control; antigenic stimulation did not induce degradation of these receptors in the various clones studied.



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  		FIGURE 5. ZAP-70 specifically controls the turnover of {zeta} induced by TCR stimulation but not the constitutive turnover of this chain. A, The various P116 clones were biotinylated and lysed, either immediately after biotinylation or after incubation for 2 h in medium alone. B, Cells were incubated for 1.5 h at 37°C in medium alone or with Raji B cells unpulsed, or pulsed for 2 h with 0.1 µg/ml SEE. Biotinylated proteins were then precipitated with streptavidin-agarose, subjected to electrophoresis, and Western blotted, and the blots were probed with an anti-{zeta}, anti-transferrin receptor (TfR). Neither {zeta} nor the transferrin receptor was precipitated by streptavidin-agarose from nonbiotinylated cells.

 
These results demonstrate that ZAP-70 is involved in the degradation of {zeta} induced by TCR activation and that this process involves the kinase activity of ZAP-70.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we investigated the role of ZAP-70 in the down-regulation of TCR/CD3 and the degradation of {zeta} induced by TCR activation. We used two different models: human T cells from patients presenting immunodeficiency due to the absence of ZAP-70 and the ZAP-70-deficient Jurkat cell clone, P116 (26). Our results demonstrate that ZAP-70 is involved in the down-modulation of CD3/TCR and the degradation of {zeta}. The expression of a kinase-dead mutant of ZAP-70 in P116 cells restored neither the TCR-induced down-modulation of CD3/TCR nor the degradation of {zeta}, demonstrating that ZAP-70 tyrosine kinase activity is required for both phenomena. Of note, the TCR down-modulation and {zeta} degradation observed in P116 transfected with wild-type ZAP-70 was always more important than in the wild-type Jurkat clone used in this study. This correlated with an overexpression of ZAP-70 in P116 ZAPwt and can be due to the fact that either ZAP-70 or its substrate(s) are limiting factors in the TCR modulation and degradation.

The results presented in this paper show that the absence of an active ZAP-70 does not affect the down-regulation of TCR or CD3{epsilon} at high concentrations of anti-CD3 Ab or superantigen. In contrast, down-modulation is reduced at low doses of these activators. These results are consistent with a study showing that PP1, a PTK inhibitor, decreases TCR down-modulation at low receptor occupancy by Ab or Ag whereas the effects of the inhibitor are undetectable at high TCR occupancy (33). This previous study suggested that the down-modulation of the TCR is promoted by both tyrosine kinase-dependent and tyrosine kinase-independent mechanisms. Our study shows that the down-regulation of the TCR at low receptor occupancy is dependent on ZAP-70. In physiological conditions, T cells are most likely to be activated in conditions of low TCR occupancy; therefore, our results are pertinent.

Previous studies have shown that tyrosine kinases are involved in TCR down-modulation and degradation. Jurkat cell mutants lacking the PTK Lck or the regulatory protein tyrosine phosphatase CD45 display less-efficient TCR down-regulation than their parental cell lines in response to anti-TCR-CD3 Abs or bacterial superantigens (15, 17). The degradation of {zeta} was also studied in the Lck-deficient mutant and was found to be impaired (15). The Lck PTK is involved in tyrosine phosphorylation of the ITAMs present in the CD3-{zeta} chains and, thus, in the recruitment of ZAP-70 (34). Moreover, Lck binds to ZAP-70 (35), thereby regulating its activity (36). Therefore, the inhibition of Lck activity also leads to ZAP-70 inhibition. The defective {zeta} degradation observed in Lck-deficient cells may result from a lack of activation of ZAP-70 because, as shown here, the kinase activity of ZAP-70 is required for TCR-induced {zeta} degradation.

Several studies have shown that the TCR/CD3 complex has a long lifetime and is recycled on resting T cells (3, 4, 5). A recent study showed that TCR activation does not increase internalization of the TCR but instead prevents TCR from recycling back to the cell surface following Ag stimulation by three processes: intracellular retention, degradation in late endosomes/lysosomes, and degradation in the cytosol by proteasomes (5). Our results suggest that, in the absence of ZAP-70 and at high TCR occupancy, {zeta} is not targeted for degradation whereas TCR is internalized because TCR down-regulation is normal. Thus, in the absence of ZAP-70 tyrosine kinase activity, {zeta} is probably retained in the endosomal pathway. In contrast, our results show that ZAP-70 does not control the constitutive degradation of {zeta} in resting T cells, suggesting that the constitutive degradation and activation-induced degradation of {zeta} are controlled by two independent mechanisms. We are currently investigating the endosomal trafficking of {zeta} in both cases.

The results presented in this paper are consistent with those of a previous study showing that Syk, a PTK from the same family as ZAP-70, is involved in the transport of the Fc{gamma}R (25). The authors showed that a point mutation in the gene encoding the immunoreceptor-associated {gamma}-chain ITAM affecting Syk activation, and overexpression of a Syk dominant negative mutant, inhibited signal transduction. These mutations did not affect internalization of the Fc{gamma}R but impaired Fc{gamma}R transport from endosomes to lysosomes. The mechanisms and the potential substrates of Syk involved in the transport of FcR to lysosomes are unknown. Our results suggest that ZAP-70 may act in a similar manner, because the degradation of {zeta}, which has been shown by others to take place in lysosomes (15, 24), requires ZAP-70.

What molecule acts as the partner of ZAP-70 in controlling TCR down-modulation and {zeta} degradation? The c-Cbl (Casitas B cell lymphoma) protooncogene binds directly to both ZAP-70 and Syk via a motif involving phosphotyrosine 292 in ZAP-70 and an orthologous docking site in Syk (37, 38, 39). The members of the Cbl family are molecular adapters, recently identified as part of the ubiquitin ligation machinery involved in the degradation of phosphorylated proteins (40, 41, 42). Ubiquitination is an important process, involving the attachment of a ubiquitin molecule to a protein, thereby targeting that protein to degradation compartments (43). For example, epidermal growth factor receptors that recruit c-Cbl after the binding of their ligands are ubiquitinated and targeted to lysosomal and proteasomal compartments, whereas receptors that did not bind c-Cbl are recycled back to the plasma membrane; therefore, c-Cbl may be involved in the endocytotic sorting of tyrosine kinase receptors (44). By targeting tyrosine kinase receptors to degradative compartments, c-Cbl functions as a negative regulator of these receptors. It has also been shown to regulate Syk and ZAP-70 kinase activity (45). Compelling evidence that mammalian c-Cbl functions as a negative regulator of tyrosine kinase activity has also been provided by studies of c-Cbl-deficient mice (46, 47). These mice present tissue hyperplasia and enhanced T cell signaling (48). In particular, {zeta} levels are much higher in the T cells of c-Cbl-/- mice than in those of their normal littermates (48). A very recent study showed that Cbl promotes ubiquitination of the {zeta}-chain and that ZAP-70, by binding to both {zeta} and c-Cbl, acts as an adapter (49). Thus, the binding of c-Cbl to ZAP-70 may regulate the ubiquitination-dependent targeting of {zeta} to degradative compartments. Our results show that, for {zeta} degradation to occur, ZAP-70 must not only be present but must also be enzymatically active. This result is not simply due to defective binding of the kinase-dead mutant ZAP-70 D461/N to {zeta}, because this molecule did bind {zeta} after TCR activation (data not shown). It also cannot be accounted for by an inability of the mutant ZAP-70 to protect {zeta} against dephosphorylation because {zeta} was phosphorylated to similar extents in P116ZAPwt and P116ZAP{Delta}K cells (Fig. 2Go). Therefore, ZAP-70 kinase activity may play a role by autophosphorylation of its Y292 residue, facilitating interaction with c-Cbl. However, this is unlikely because CD3 activation induces the coimmunoprecipitation of c-Cbl with the kinase-dead ZAP-70 used in this study (data not shown). Alternatively, it may modify c-Cbl activity by direct phosphorylation of c-Cbl or phosphorylation of its downstream effectors. We are currently testing these hypotheses.

The export of TCR/CD3 complexes from the endoplasmic reticulum and their transport to the cell surface require the concomitant production and assembly of the {alpha}{beta} chains of the TCR and the {gamma}{epsilon} and {delta}{epsilon} chains of the CD3, as well as {zeta}-{zeta} dimers. However, the half-lives of the various subunits differ greatly: >20 h for the CD3 complex (8) and <4 h for the {zeta}-chain (32). This suggests that the various subunits segregate at the plasma membrane or during intracellular trafficking. Some subunits are internalized and recycled back to the cell surface, whereas others are sorted for degradation. {zeta} has been shown to contain an internalization motif, as chimeras containing the cytosolic domain of {zeta} are internalized upon cross-linking of the chimeras (33). Therefore, {zeta} may be internalized and sorted independently of TCR/CD3. The control of {zeta} degradation by ZAP-70 may be unique to this chain and we are currently investigating whether degradation of the other chains of the CD3/TCR complex is also controlled by ZAP-70.

In conclusion, our results show that ZAP-70, an essential factor for T cell activation, also controls the termination of T cell activation and that the kinase activity of ZAP-70 is required for this process. This control of two antinomic phenomena by the same molecule must be tightly regulated in time and space but may be a highly effective means of preventing T cell hyperresponsiveness.


   Acknowledgments
 
We thank Drs. A. Alcover and S. Amigorena for critical reading of the manuscript and helpful discussions. We also thank Françoise Le Deist for assistance in the characterization of the ZAP-70-deficient patients.


   Footnotes
 
1 This work was supported by institutional grants from Institut National de la Santé et de la Recherche Médicale and specific grants from the Association pour la Recherche sur le Cancer. C.D. holds an Association pour la Recherche sur le Cancer fellowship. Back

2 Address correspondence and reprint requests to Dr. Claire Hivroz, Institut National de la Santé et de la Recherche Médicale, Unité 520, Institut Curie, 12 Rue Lhomond, 75005 Paris, France. E-mail address: claire.hivroz{at}curie.fr Back

3 Abbreviations used in this paper: PTK, protein tyrosine kinase; ITAM, immunoreceptor tyrosine-based activation motif; Erk, extracellular signal-regulated kinase; SEE, staphylococcal enterotoxin E; MAPK, mitogen-activated protein kinase; MFI, mean fluorescence intensity. Back

Received for publication January 16, 2002. Accepted for publication June 4, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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